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DECREASED PCSK9 FUNCTION INCREASES ENDOTOXIN CLEARANCE AND DECREASES THE ASSOCIATED INFLAMMATORY RESPONSE
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513 ABSENCE OF FOUR-AND-A-HALF LIM DOMAIN PROTEIN 2 DECREASES ATHEROSCLEROSIS IN APOE-/- MICE: ROLE OF MONOCYTIC IMMUNE CELLS T Ebrahimian, D Simon, CA Lemarié, S Simeone, M Heidari, KK Mann, S Wassmann, S Lehoux Montréal, Québec BACKGROUND:
Four-and-a-half LIM domain protein-2 (FHL2) is expressed in endothelial and vascular smooth muscle cells. It negatively regulates endothelial cell survival and migration, but its role in atherogenesis is unknown. METHODS AND RESULTS: To investigate the role of FHL2 in atherosclerosis, FHL2-deficient (FHL2-/-) mice were crossed with ApoE-deficient (ApoE -/-) mice, to generate ApoE/FHL2-/- mice. After 7 weeks of high fat diet, ApoE/FHL2-/- mice had significantly smaller (P<0.05) atherosclerotic plaques than ApoE-/- mice in the aortic sinus (0.140.02 vs. 0.290.04 mm2), the brachiocephalic artery (0.030.008 vs. 0.070.01 mm2) and the aorta (6.90.9 vs 10.31%), assessed by oil red O staining. This was associated with enhanced collagen (162 vs 8.63 %) and smooth muscle cell (4.50.8 vs 1.80.5%) contents within the plaques of ApoE/FHL2-/- mice. Moreover, macrophage content within plaques was reduced 2-fold in ApoE/FHL2-/- vs ApoE-/- mice (P<0.05). This could be explained, in part, by the 40% reduction in aortic ICAM-1 mRNA and 55% reduction in VCAM-1 protein expression in the plaque. Furthermore, aortic gene expression of Cx3cl1 and Ccl5 was increased in ApoE/FHL2-/- vs ApoE-/- mice, suggesting that different monocyte populations might be recruited to the plaques. Peritoneal thioglycollate injection elicited equivalent numbers of monocytes and macrophages in both groups, but a significantly lower number of pro-inflammatory Ly6C high monocytes were recruited in ApoE/FHL2-/- vs ApoE-/- mice (83 vs 184%). Furthermore, mRNA levels of Cx3cr1 were 2-fold higher in monocytes from ApoE/FHL2-/- mice compared with ApoE-/mice. Finally, we also investigated the potential importance of myeloid cell FHL2 deficiency in atherosclerosis. ApoE-/- or ApoE/ FHL2-/- mice were lethally irradiated and transplanted with ApoE-/or ApoE/FHL2-/- bone marrow. After recovery and 7 weeks of high fat diet, both chimeric groups developed smaller plaques than ApoE-/- mice transplanted with ApoE-/- bone marrow. CONCLUSION: These results suggest that FHL2 in both myeloid and vascular cells may play an important role in atherosclerosis by promoting pro-inflammatory chemokine production, adhesion molecule expression, and pro-inflammatory monocyte recruitment. 514 DECREASED PCSK9 FUNCTION INCREASES ENDOTOXIN CLEARANCE AND DECREASES THE ASSOCIATED INFLAMMATORY RESPONSE MS Cirstea, JH Boyd, KR Thain, KR Walley Vancouver, British Columbia BACKGROUND:
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating enzyme that targets the low density lipoprotein receptor (LDLR) for degradation. Agents inhibiting
Canadian Journal of Cardiology Volume 30 2014
PCSK9 thus result in higher hepatocyte LDLR density and increased LDL clearance from the circulation. Endotoxins are also lipid moieties that can associate with LDL and may be cleared via the LDL receptor. Here, we hypothesize that the bacterial endotoxin lipopolysaccharide (LPS) is cleared via LDLR, and that decreased PCSK9 function will increase the rate of LPS clearance resulting in a decrease in the cytokine inflammatory response. Since atherosclerotic vascular disease also involves an inflammatory component, decreased PCSK9 function may have additional important anti-inflammatory effects. METHODS AND RESULTS: LPS (10 mg/kg) was injected intraperitoneally into wildtype and Pcsk9 -/- mice. After 6h, Pcsk9 -/- mice showed decreased circulating LPS (implying increased LPS clearance), decreased pro-inflammatory cytokine levels, and more normal temperature and activity compared to WT controls. To test whether these observations result from the interplay between PCSK9, LPS and the LDLR, HepG2 (immortalized hepatocyte) cells grown in media containing recombinant human PCSK9 were pretreated either with anti-PCSK9 antibody or isotype control, and then with fluorescently conjugated LPS. After 24h, flow cytometry was used to measure both LPS uptake and LDLR receptor density. Cells treated with the anti-PCSK9 antibody showed both elevated cell surface LDLR levels, and increased fluorescent LPS internalization. CONCLUSION: Decreased PCSK9 function due to gene knockout or due to PCSK9 inhibition results in increased LPS clearance in vivo and increased LDLR density and LPS uptake in cultured hepatocytes in vitro. These results suggest that PCSK9 inhibition may have a novel and important role to play in modulating the inflammatory response by promoting increased clearance of proinflammatory lipid based molecules such as LPS. We postulate that this additional effect, beyond the direct LDL lowering effect, may contribute to beneficial clinical outcomes in treating the inflammatory aspects of atherosclerotic vascular disease. 515 PROGRESSIVE AORTIC DILATATION IS REGULATED BY THE MIR17 CLUSTER J Wu, J Guo, S Li, K Tsang, L Tumiati, C David, J Bos, M Ouzounian, TM Yau, TE David, RD Weisel, R Li Toronto, Ontario BACKGROUND:
Ascending aortic aneurysms are characterized by progressive dilatation due to activation of matrix metaloproteinases (MMPs), matrix disruption and loss of elasticity. Emerging evidence suggests that microRNAs (miRNAs) may contribute to the pathogenesis of aortic dilatation, but the exact miRNAs responsible have not been determined. The goal of this investigation was to identify the miRNAs responsible for progressive aortic dilation in patients with Marfan syndrome and bicuspid aortic valve (BAV) aortopathy. METHODS AND RESULTS: Aortic tissue samples (approximately 11cm) were collected from the dilated aneurysmal segment and adjacent nondilated segments which appeared normal at aortic surgery. The nondilated was compared to the dilated samples to eliminate differences due to individual patient variations. The nondilated segments represent the early stages of aortic matrix
513 ABSENCE OF FOUR-AND-A-HALF LIM DOMAIN PROTEIN 2 DECREASES ATHEROSCLEROSIS IN APOE-/- MICE: ROLE OF MONOCYTIC IMMUNE CELLS T Ebrahimian, D Simon, CA Lemarié, S Simeone, M Heidari, KK Mann, S Wassmann, S Lehoux Montréal, Québec BACKGROUND:
Four-and-a-half LIM domain protein-2 (FHL2) is expressed in endothelial and vascular smooth muscle cells. It negatively regulates endothelial cell survival and migration, but its role in atherogenesis is unknown. METHODS AND RESULTS: To investigate the role of FHL2 in atherosclerosis, FHL2-deficient (FHL2-/-) mice were crossed with ApoE-deficient (ApoE -/-) mice, to generate ApoE/FHL2-/- mice. After 7 weeks of high fat diet, ApoE/FHL2-/- mice had significantly smaller (P<0.05) atherosclerotic plaques than ApoE-/- mice in the aortic sinus (0.140.02 vs. 0.290.04 mm2), the brachiocephalic artery (0.030.008 vs. 0.070.01 mm2) and the aorta (6.90.9 vs 10.31%), assessed by oil red O staining. This was associated with enhanced collagen (162 vs 8.63 %) and smooth muscle cell (4.50.8 vs 1.80.5%) contents within the plaques of ApoE/FHL2-/- mice. Moreover, macrophage content within plaques was reduced 2-fold in ApoE/FHL2-/- vs ApoE-/- mice (P<0.05). This could be explained, in part, by the 40% reduction in aortic ICAM-1 mRNA and 55% reduction in VCAM-1 protein expression in the plaque. Furthermore, aortic gene expression of Cx3cl1 and Ccl5 was increased in ApoE/FHL2-/- vs ApoE-/- mice, suggesting that different monocyte populations might be recruited to the plaques. Peritoneal thioglycollate injection elicited equivalent numbers of monocytes and macrophages in both groups, but a significantly lower number of pro-inflammatory Ly6C high monocytes were recruited in ApoE/FHL2-/- vs ApoE-/- mice (83 vs 184%). Furthermore, mRNA levels of Cx3cr1 were 2-fold higher in monocytes from ApoE/FHL2-/- mice compared with ApoE-/mice. Finally, we also investigated the potential importance of myeloid cell FHL2 deficiency in atherosclerosis. ApoE-/- or ApoE/ FHL2-/- mice were lethally irradiated and transplanted with ApoE-/or ApoE/FHL2-/- bone marrow. After recovery and 7 weeks of high fat diet, both chimeric groups developed smaller plaques than ApoE-/- mice transplanted with ApoE-/- bone marrow. CONCLUSION: These results suggest that FHL2 in both myeloid and vascular cells may play an important role in atherosclerosis by promoting pro-inflammatory chemokine production, adhesion molecule expression, and pro-inflammatory monocyte recruitment. 514 DECREASED PCSK9 FUNCTION INCREASES ENDOTOXIN CLEARANCE AND DECREASES THE ASSOCIATED INFLAMMATORY RESPONSE MS Cirstea, JH Boyd, KR Thain, KR Walley Vancouver, British Columbia BACKGROUND:
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating enzyme that targets the low density lipoprotein receptor (LDLR) for degradation. Agents inhibiting
Canadian Journal of Cardiology Volume 30 2014
PCSK9 thus result in higher hepatocyte LDLR density and increased LDL clearance from the circulation. Endotoxins are also lipid moieties that can associate with LDL and may be cleared via the LDL receptor. Here, we hypothesize that the bacterial endotoxin lipopolysaccharide (LPS) is cleared via LDLR, and that decreased PCSK9 function will increase the rate of LPS clearance resulting in a decrease in the cytokine inflammatory response. Since atherosclerotic vascular disease also involves an inflammatory component, decreased PCSK9 function may have additional important anti-inflammatory effects. METHODS AND RESULTS: LPS (10 mg/kg) was injected intraperitoneally into wildtype and Pcsk9 -/- mice. After 6h, Pcsk9 -/- mice showed decreased circulating LPS (implying increased LPS clearance), decreased pro-inflammatory cytokine levels, and more normal temperature and activity compared to WT controls. To test whether these observations result from the interplay between PCSK9, LPS and the LDLR, HepG2 (immortalized hepatocyte) cells grown in media containing recombinant human PCSK9 were pretreated either with anti-PCSK9 antibody or isotype control, and then with fluorescently conjugated LPS. After 24h, flow cytometry was used to measure both LPS uptake and LDLR receptor density. Cells treated with the anti-PCSK9 antibody showed both elevated cell surface LDLR levels, and increased fluorescent LPS internalization. CONCLUSION: Decreased PCSK9 function due to gene knockout or due to PCSK9 inhibition results in increased LPS clearance in vivo and increased LDLR density and LPS uptake in cultured hepatocytes in vitro. These results suggest that PCSK9 inhibition may have a novel and important role to play in modulating the inflammatory response by promoting increased clearance of proinflammatory lipid based molecules such as LPS. We postulate that this additional effect, beyond the direct LDL lowering effect, may contribute to beneficial clinical outcomes in treating the inflammatory aspects of atherosclerotic vascular disease. 515 PROGRESSIVE AORTIC DILATATION IS REGULATED BY THE MIR17 CLUSTER J Wu, J Guo, S Li, K Tsang, L Tumiati, C David, J Bos, M Ouzounian, TM Yau, TE David, RD Weisel, R Li Toronto, Ontario BACKGROUND:
Ascending aortic aneurysms are characterized by progressive dilatation due to activation of matrix metaloproteinases (MMPs), matrix disruption and loss of elasticity. Emerging evidence suggests that microRNAs (miRNAs) may contribute to the pathogenesis of aortic dilatation, but the exact miRNAs responsible have not been determined. The goal of this investigation was to identify the miRNAs responsible for progressive aortic dilation in patients with Marfan syndrome and bicuspid aortic valve (BAV) aortopathy. METHODS AND RESULTS: Aortic tissue samples (approximately 11cm) were collected from the dilated aneurysmal segment and adjacent nondilated segments which appeared normal at aortic surgery. The nondilated was compared to the dilated samples to eliminate differences due to individual patient variations. The nondilated segments represent the early stages of aortic matrix